US2003077645A1PendingUtilityA1
Superfamily receptor chimeras, translocation assay for superfamily receptor ligands, and methods and kits for detecting and characterizing receptor ligands
Priority: Sep 28, 2001Filed: Sep 30, 2002Published: Apr 24, 2003
Est. expirySep 28, 2021(expired)· nominal 20-yr term from priority
G01N 33/6875C12N 15/1027C07K 14/721C07K 2319/00A61K 38/00
38
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Claims
Abstract
The present invention relates to novel superfamily receptor chimeras, methods for using superfamily receptor chimeras, methods and kits for detecting cellular function and metabolic state, and the treatment of disease states involving defective receptor protein cytoplasm/nuclear translocation. In particular, this invention is preferably directed to chimeras of glucocorticoid receptor/superfamily receptor proteins.
Claims
exact text as granted — not AI-modifiedWe claim:
1 . A method for making a recombinant nuclear translocation protein, comprising:
covalently connecting (i) a glucocorticoid receptor DNA sequence coding for the cytoplasmic/nuclear translocation domain of the glucocorticoid receptor protein, (ii) a superfamily receptor DNA sequence coding for the ligand binding domain of a superfamily receptor protein, and (iii) a nucleic acid sequence for a marker protein domain, to form a DNA chimera,
wherein said superfamily receptor DNA sequence is connected to the 3′ end of said glucocorticoid receptor DNA sequence; and
expressing said DNA chimera in an expression system to prepare said protein.
2 . The method of claim 1 , further comprising:
covalently connecting said translocation domain of the glucocorticoid receptor and said ligand binding domain of a superfamily receptor utilizing a DNA linker sequence.
3 . The method of claim 2 , wherein said DNA linker sequence is a fragment of a polylinker sequence.
4 . The method of claim 1 , wherein said marker protein domain DNA sequence is covalently connected to the 5′ end of said glucocorticoid receptor DNA sequence.
5 . The method of claim 4 , further comprising:
covalently connecting said glucocorticoid receptor DNA sequence and said nucleic acid sequence for a marker protein utilizing a DNA linker sequence.
6 . The method of claim 1 , wherein said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including the complete nuclear localization sequence of said glucocorticoid receptor DNA sequence.
7 . The method of claim 1 , wherein said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including the complete nuclear localization sequence and the complete helix 1 sequence of said glucocorticoid receptor DNA sequence.
8 . The method of claim 1 , wherein said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including DNA bases corresponding to about amino acid residue 570 of said glucocorticoid receptor protein.
9 . The method of claim 1 , wherein said superfamily receptor DNA sequence encompasses the 3′ end of said sequence through and including the complete ligand binding domain sequence and the complete helix 3 sequence of said superfamily receptor DNA sequence.
10 . The method of claim 1 , wherein said superfamily receptor DNA sequence encompasses the 3′ end of said sequence through and including the complete ligand binding domain sequence, the complete helix 3 sequence, and at most a fragment of helix 1 of said superfamily receptor DNA sequence.
11 . A protein produced by the process of:
covalently connecting (i) a glucocorticoid receptor DNA sequence coding for the 5′ end of the DNA sequence of the glucocorticoid receptor protein, through and including the complete nuclear localization sequence and the complete helix 1 sequence of said glucocorticoid receptor DNA sequence, (ii) a superfamily receptor DNA sequence coding for the 3′ end of the DNA sequence of a superfamily receptor protein, through and including the complete ligand binding domain sequence and the complete helix 3 sequence of said superfamily receptor DNA sequence, and (iii) a nucleic acid sequence for a marker protein domain, to form a DNA chimera,
wherein said superfamily receptor DNA sequence is connected to the 3′ end of said glucocorticoid receptor DNA sequence,
wherein said marker protein domain DNA sequence is covalently connected to the 5′ end of said glucocorticoid receptor DNA sequence,
and wherein said translocation domain of the glucocorticoid receptor and said ligand binding domain of a superfamily receptor are covalently connected by a DNA linker sequence; and
expressing said DNA chimera in an expression system to prepare said protein.
12 . A nucleic acid chimera comprising:
a nucleic acid sequence which codes for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein; and a nucleic acid sequence which codes for the ligand binding domain of a superfamily receptor protein.
13 . The nucleic acid chimera of claim 12 , additionally comprising:
a nucleic acid sequence for a marker protein domain.
14 . The nucleic acid chimera of claim 13 , wherein said marker protein domain encodes a fluorescent protein.
15 . The nucleic acid chimera of claim 12 , wherein said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including the complete nuclear localization sequence of said glucocorticoid receptor DNA sequence.
16 . The nucleic acid chimera of claim 12 , wherein said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including the complete nuclear localization sequence and the complete helix 1 sequence of said glucocorticoid receptor DNA sequence.
17 . The nucleic acid chimera of claim 12 , wherein said glucocorticoid receptor DNA sequence encompasses the 5′ end of said sequence through and including DNA bases corresponding to about amino acid residue 570 of said glucocorticoid receptor protein.
18 . The nucleic acid chimera of claim 12 , wherein said superfamily receptor DNA sequence encompasses the 3′ end of said sequence through and including the complete ligand binding domain sequence and the complete helix 3 sequence of said superfamily receptor DNA sequence.
19 . The nucleic acid chimera of claim 12 , wherein said superfamily receptor DNA sequence encompasses the 3′ end of said sequence through and including the complete ligand binding domain sequence, the complete helix 3 sequence, and at most a fragment of helix 1 of said superfamily receptor DNA sequence.
20 . The nucleic acid chimera of claim 12 , wherein said ligand binding domain is the ligand binding domain of estrogen receptor.
21 . The nucleic acid chimera of claim 12 , which is SEQ. ID NO. 1.
22 . The nucleic acid chimera of claim 12 , wherein said ligand binding domain is the ligand binding domain of retinoic acid receptor.
23 . The nucleic acid chimera of claim 12 , which is SEQ. ID NO. 2.
24 . A chimeric protein comprising two elements:
a glucocorticoid receptor 5′ end, encompassing the nuclear translocation domain and helix 1; and a superfamily receptor 3′ end, encompassing the ligand binding domain and helix 3.
25 . The chimeric protein of claim 24 , further comprising a marker protein domain.
26 . The chimeric protein of claim 25 , wherein said marker protein domain encodes a fluorescent protein.
27 . The chimeric protein of claim 24 , wherein said ligand binding domain is the ligand binding domain of estrogen receptor.
28 . The chimeric protein of claim 24 , which is SEQ. ID NO. 3.
29 . The chimeric protein of claim 24 , wherein said ligand binding domain is the ligand binding domain of retinoic acid receptor.
30 . The chimeric protein of claim 24 , which is SEQ. ID NO. 4.
31 . A method for detecting a ligand of a superfamily receptor protein, which comprises:
producing a nucleic acid vector encoding a nucleic acid chimera comprising three elements: a 5′ end of a glucocorticoid receptor, encompassing the nuclear translocation domain and helix 1, a 3′ end of a superfamily receptor, encompassing the ligand binding domain and helix 3, and a nucleic acid sequence for a marker protein domain; transfecting a eukaryotic cell with said nucleic acid vector; isolating a clonal population of cells that express a chimeric protein translated from said nucleic acid vector; contacting said cells with a sample compound or composition; and detecting the presence of cytoplasmic/nuclear translocation in response to a ligand of said ligand binding domain.
32 . The method of claim 31 , wherein said marker protein domain encodes a fluorescent protein.
33 . A method for determining the concentration of a ligand of a labeled chimeric superfamily receptor protein, which comprises:
producing a nucleic acid vector encoding a nucleic acid chimera comprising: a nucleic acid sequence coding for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, a nucleic acid sequence coding for the ligand binding domain of a superfamily receptor protein, and a nucleic acid sequence for a marker protein domain; transfecting a eukaryotic cell with said nucleic acid vector; isolating a clonal population of transfected cells that express a chimeric protein translated from said nucleic acid vector; contacting said transfected cells with a sample; scanning one or more test cell(s) to obtain signal data from said labeled protein; converting said signal data to obtain the cellular location of said labeled protein in said test cell(s); and analyzing said data using an analysis system having an algorithm to calculate changes in the distribution of said labeled protein between the cell cytoplasm and the cell nucleus of said test cell(s), said analysis system having the capability of providing an accurate reading of the concentration of a ligand.
34 . The method of claim 33 , wherein said marker protein domain encodes a fluorescent protein.
35 . A kit for detecting and screening for a ligand of a superfamily receptor protein in an environmental sample, comprising:
a cell-based system which expresses a chimeric protein comprising the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, the ligand binding domain of a superfamily receptor protein, and a marker protein domain; and a detection system for the detection of the translocation of said marker protein.
36 . The kit of claim 35 , additionally comprising:
one or more compounds and/or compositions which stably associate with said chimeric protein in the absence of a ligand for the ligand binding domain of said chimeric protein, and which dissociates from said chimeric protein in the presence of a ligand for the ligand binding domain of said chimeric protein.
37 . A kit for detecting and screening for a ligand of a superfamily receptor protein in an environmental sample, comprising:
a quantity of a chimeric protein comprising the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, the ligand binding domain of a superfamily receptor protein, and a marker protein domain; a cell-free membrane system which restricts translocation of the chimeric protein when no ligand is bound to the ligand binding domain of said chimeric protein, and which permits translocation of the chimeric protein when the ligand binding domain of said chimeric protein is bound to its ligand; and a detection system for the detection of the translocation of said marker protein.
38 . The kit of claim 37 , additionally comprising:
one or more compounds and/or compositions which stably associate with said chimeric protein in the absence of a ligand for the ligand binding domain of said chimeric protein, and which dissociates from said chimeric protein in the presence of a ligand for the ligand binding domain of said chimeric protein.
39 . A method for diagnosis of defects in the nuclear transportation process, which comprises:
producing a nucleic acid vector encoding a nucleic acid chimera comprising: a nucleic acid sequence coding for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein, a nucleic acid sequence coding for the ligand binding domain of a superfamily receptor protein, and a nucleic acid sequence for a marker protein domain; transfecting a set of suspected defective cells with said nucleic acid vector; isolating a clonal population of said cells that express a chimeric protein translated from said nucleic acid vector; contacting said cells with a ligand of said ligand binding domain; and detecting the presence or absence of cytoplasmic/nuclear translocation in response to said ligand.
40 . A method for treating defective translocation of a superfamily receptor protein from the cytoplasm to the nucleus of a cell, in an animal in need thereof, comprising:
producing a nucleic acid vector which is capable of being transcribed, and which encodes a nucleic acid chimera comprising: a nucleic acid sequence coding for the cytoplasmic/nuclear translocation domain of glucocorticoid receptor protein and a nucleic acid sequence coding for the ligand binding domain of said superfamily receptor protein; and transfecting a target cell in said animal with said nucleic acid vector.
41 . A pharmaceutical composition comprising:
(i) a chimeric protein comprising two elements:
a glucocorticoid receptor 5′ end, encompassing the nuclear translocation domain and helix 1; and
a superfamily receptor 3′ end, encompassing the ligand binding domain and helix 3; and
(ii) a pharmaceutically acceptable carrier.Join the waitlist — get patent alerts
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